Gammadelta T cells are found in a variety of vertebrate animals. Despite intense efforts, their role in the immune system has remained elusive. While they are clearly dominant in a few pathological situations, their failure to be significantly involved in most immune responses suggests that they perform certain functions uniquely and efficiently and thus may contribute to host immune defense systems differently than alphabeta T cells. In order to better understand how gammadelta T cells contribute to immunological competence, the rules governing their recognition need to be understood. In particular, while most alphabeta T cells recognize processed antigens in the context of a major histocompatibility gene complex (MHC) molecule, many specific gammadelta T cells have no demonstratable MHC restriction. In addition, although there are gammadelta T cells with allospecificity to MHC or MHC-like molecules, many aspects of their specificity suggest a fundamental difference between gammadelta T cell and alphabeta T cells. The object of the research described here is to clarify these issues by analyzing in detail the precise nature of the specificity of three different alloreactive gammadelta T cell clones. Our goal is to understand: (i) When gammadelta T cell recognition involves a MHC molecule, whether or not the MHC molecule plays a similar role as in alphabeta T cell recognition, (ii) if and what type of antigen processing is required, (iii) which part of the MHC molecule is being recognized, and, if a peptide/MHC complex is recognized, what is the nature of the peptide, (iv) how general these rules are. Our initial focus is on the Class II MHC allo-reactive gammadelta T cell, LBK-5, which is I-Ek,b,s reactive. We will first analyze LBK-5 TCR/I-Ek interactions with antigen presenting cells (APC) expressing mutant I-Ek molecules, and the antigen processing requirements will be probed with APCs defective in the endocytic pathway. Our preliminary results suggest that LBK-5 interacts with I-Ek very differently than any of the alphabeta T cells tested, and that it has distinct antigen processing requirements as well. These experiments will be confirmed and extended, and used as a basis to identify the ligand(s). The generality of these results will be examined by parallel studies of other alloreactive gammadelta T cell clones, such as LKD-1 (I-Ad specific) and G8 (TLb specific). A clear understanding of how gammadelta T cells recognize these well studied molecules will provide insight into the nature of gammadelta T cell specificity and help to better define their role in the immune system.